The existence of peptide containing immunoreactivity to bombesin has been identified in the whey of bovine milk. This is a new, previously unidentified peptide in mammalian tissue that is larger than the hepatacosal gastrin releasing peptide (GRP) from porcine non-antral mucosa and the amphibian bombesin tetradecapeptide. This "milk bombesin" was separated from GRP by both gel filtration and reverse-phase HPLC. However, both porcine GRP and chicken GRP (mammalian analogue of alytesin, another bombesin-related peptide) produced nonparallel displacement curves, whereas "milk bombesin" was always parallel. Cleavage by trypsin produced a smaller immunoreactive peptide; alkylation of a urea/DTT treated sample of "milk bombesin" had no effect on its elution pattern of gel chromatography; thus, it is neither an aggregate of GRP or bombesin or both, nor combined with any extraneous milk protein. High speed centrifugation proved that the peptide was not associated with cell debris, bacterial or viral particles, or milk lipids. These studies provide evidence for the presence of another unique form of bombesin in mamalian tissue.